Immunotherapies targeting various protein aggregates such as amyloid-? (A?), tau and ?-synuclein (?syn) are in different stages of clinical development, and collectively are the most common approach by the pharmaceutical industry to tackle diseases characterized by such depositions [1-3]. The majority of these potential therapies are whole antibodies. Much less attention has been paid to antibody fragments, which have certain advantages and their therapeutic and diagnostic potential should be explored further. Specifically, single domain antibodies (sdAbs) are of particular interest because their small size (13 kDa) improves tissue penetration, including through the blood-brain-barrier, allowing better access over antibodies (150 kDa) to the target molecule and its hidden epitopes. Importantly as well, they are high affinity, and easy to engineer and grow in large quantities. The Specific Aim is to determine the therapeutic potential of camelid single-domain heavy chain antibody fragments (sdAbs) against the ?syn protein. It is hypothesized that their small size will provide therapeutic benefits over whole antibodies, primarily because of greater access to the target, and to some extent due to their binding to novel epitopes that the larger antibodies cannot access. We have generated 51 clones of sdAbs, with unique complementary determining regions (CDRs), that recognize various forms of ?syn and propose to determine their therapeutic potential in different models, as well as to clarify their mechanism of action. For the initial screen, up to 30 of the individual sdAbs will be expressed globally in neurons of fruit fly models of synucleinopathy, and their ability to prevent/attenuate the ?syn pathology monitored. Subsequently, the 5 most efficacious sdAbs will be examined further in flies by expressing them in astrocytes or peripherally, and in cell culture models of synucleinopathy for mechanistic insight, and the most effective one from that study in an animal model. Our preliminary data support the feasibility of screening for therapeutic efficacy of antibody fragments targeting the tau protein in fly models. Specifically, we show that neuronal expression of an anti-tau single chain variable antibody fragment (scFv) prevents developmental toxicity of overexpression of human tau with or without a tauopathy mutation, and significantly extends the life span of the tauopathy flies. We expect that expressing sdAbs against ?syn in synucleinopathy flies will also provide therapeutic benefits. The proposed studies may identify a novel class of therapy candidates for Lewy Body Dementia, Parkinson's disease, Alzheimer's disease with Lewy Bodies, Multiple System Atrophy and for other synucleinopathies, with direct relevance to various other protein misfolding disorders.